The importance of PKC? in TCR signals controlling T cell activation and differentiation is well established. The selective requirement of PKC? in allergic and autoimmune diseases, but not in antiviral responses, highlights it as a promising drug target. Realization of this potential requires a clear understanding of the mechanism(s) that regulate the unique, non-redundant functions and highly selective immunological synapse (IS) localization of PKC? in T cells, which remain unknown. Our work during the last funding period revealed that within the large PKC family, PKC? represents a unique paradigm with respect to the mechanisms that regulate these events and, ultimately, lead to the activation of critical transcription factors, which determine T cell fate. We propose to apply a multi-disciplinary approach in order to address the following unresolved questions: Aim 1) Positive regulation of PKC? activation and localization. Using biochemical, genetic, imaging and biophysical approaches, we will address the hypothesis that the localization and activation of PKC? in T cells are positively regulated by specialized mechanisms and plasma membrane interactions owing to the unique properties of its regulatory C1 and V3 domains. We will conduct a detailed structure- function analysis of the C1A, C1B and V3 domains of PKC?, their phosphorylation, and their interaction with binding partners. Aim 2) Autoinhibition by the PKC? C2 domain. Our studies indicate that the PKC? N- terminal C2 domain negatively regulates its function, and that autoinhibition is relieved by TCR-induced phosphorylation of Tyr-90, and, furthermore, that C2 itself is a pTyr- and PIP2-binding domain. We will investigate the mechanisms through which autoinhibition is effected, and characterize C2-binding ligands and their functional significance. We will pursue Aims 1 and 2 as a collaborative consortium with Dr. Wonhwa Cho (U. Illinois), who will provide his expertise in biophysical and two-photon imaging approaches to further elucidate the specialized mechanisms that regulate PKC?. Aim 3) Regulation of Ca2+/NFAT signaling by PKC?. PKC?-/- T cells display a Ca2+/NFAT signaling defect, but the mechanism linking PKC? to Ca2+ signaling is unknown. Based on our recent findings, and given the important role of Ca2+ signaling in determining the balance between productive T cell activation and anergy/apoptosis, we will characterize this pathway. Specifically, we will address the hypothesis that PKC? mediates a positive feedback loop that promotes PLC31 activation and, hence, Ca2+/NFAT signaling, via its functional and/or physical association with Tec family tyrosine kinase(s). Our studies will elucidate novel mechanisms that control the unique membrane localization and function of PKC? in T cells. The new information will likely provide a rational basis for using PKC? as a drug target in autoimmune and allergic diseases, and in T cell malignancies. PUBLIC HEALTH RELEVANCE: Protein kinase C-8 (PKC?), an enzyme expressed in T lymphocytes of the immune system, is important for the activation and survival of these cells and their ability to mount many beneficial as well as pathological immune responses, e.g., protection against certain infections, and autoimmune and allergic diseases. However, the mechanisms, which control the unique functions and intracellular localization of PKC? in T lymphocytes (which are not shared by other related members of the PKC family), are largely unknown. Here we propose to use a complementary set of biochemical, genetic, imaging and biophysical approaches in order to elucidate these mechanisms and the regulatory interactions of PKC? with other proteins and membrane lipids. Understanding of these pathways is likely to establish a more rational basis for the use of PKC? as a drug target in autoimmune and allergic diseases, and in T cell leukemias/lymphomas